SNAREs and methods of controlling cytokinesis

ABSTRACT

Two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, have been found to be important to cytokinesis in mammalian cells. Inhibition of syntaxin 2 and endobrevin/VAMP-8 function by over-expression of non-membrane anchored mutants of these proteins causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission is prevented by over-expression of these non-membrane anchored mutants, and that other cellular events preceeding midbody abscission, such as furrowing, are unaffected.

CROSS REFERENCES

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/355,323, filed Feb. 8, 2002, the contents of which areincorporated herein by reference in its entirety.

BACKGROUND TO THE INVENTION

[0002] Cytokinesis, the division of a cell into two daughter cells is afundamental process in biology common to all organisms. In animal cells,cytokinesis is a multi-step process that involves the assembly of anactin/myosin-dependent contractile ring that guides the invagination ofthe plasma membrane leading to the formation of a cleavage furrow.Furrowing proceeds until the cytoplasm is constricted to a narrowbridge, termed the midbody, that contains the remnants of the spindlemicrotubules and connects the two prospective daughter cells. Theterminal step of cytokinesis is the abscission of the midbody whichleads to completely separate daughter cells. The mechanism by whichmidbody abscission is achieved remains unknown.

[0003] So far, requirements for membrane fusion events duringcytokinesis have been identified only for stages that precede midbodyabscission. For example, exocytosis supplies the necessary additionalsurface area used for furrow ingression during cytokinesis in C. elegansand sea urchin embryos (Jantsch-Plunger and Glotzer, 1999; Shuster andBurgess, 2002). In C. elegans, furrow ingression appears to requireSyn-4, a syntaxin family member of the SNARE membrane fusion machinery(Jantsch-Plunger and Glotzer, 1999). The function of Syn-4 may not berestricted to furrow ingression, however, because its disruption alsocauses defects in nuclear envelope reformation. Rab3, a member of afamily of proteins implicated in the regulation of SNARE function, hasbeen implicated in furrow ingression in sea urchin embryos but it mayalso act at earlier stages because its disruption also leads to failureof nuclear division (Conner and Wessel, 2000). SNARE proteins such assyntaxin have been implicated previously in cell division in sea urchinembryos.

[0004] Cell division is a highly coordinated event requiring a varietyof membrane fusion and fragmentation events. During mitosis in highereukaryotes, for example, the nuclear envelope breaks down into nuclearmembrane vesicles after chromosome condensation, and large cytoplasmicorganelles such as the Golgi and endoplasmic reticulum (ER) are alsobelieved to fragment. These fragmented organelle membranes thendistribute equally into daughter cells and must refuse with each otherto reconstitute their respective organelles. In addition to thebreakdown and reformation of the nuclear envelope, Golgi, and ER duringthe cell cycle, the cell also increases its membrane surface area duringcell division. The final step in cell division (cytokinesis) inmammalian cells is the cleavage of a narrow cytoplasmic bridge betweenthe two daughter cells, the so called midbody.

[0005] Interestingly, recent experiments in C. elegans have indicatedthat midbody abscission can be inhibited using the fungal metabolitebrefeldin A—a drug that disrupts several organelles and traffickingpathways—under conditions that do not affect furrow ingression (Skop etal., 2001). This suggested that different cellular machineries maycontrol fusion events that facilitate furrow ingression and midbodyabscission.

SUMMARY

[0006] Two members of the SNARE membrane fusion machinery, syntaxin 2and endobrevin/VAMP-8, have been identified as being important tocytokinesis in mammalian cells. In embodiments of the present invention,functional inhibition of these proteins causes failure of midbodyabscission while earlier steps of cytokinesis are unaffected. Theseresults indicate that the terminal step of cytokinesis is not a passive“ripping-apart” or “pinching-off” mechanism but is regulated by aSNARE-mediated membrane fusion event which is distinct from exocyticevents that are involved in prior ingression of the plasma membrane.

[0007] The terminal step of cytokinesis in animal cells is theabscission of the midbody, a cytoplasmic bridge that connects the twoprospective daughter cells. Two members of the SNARE membrane fusionmachinery, syntaxin 2 and endobrevin/VAMP-8 appear near the midbodyduring cytokinesis in mammalian cells. Inhibition of syntaxin 2 andendobrevin/VAMP-8 function by over-expression of non-membrane anchoredmutants of these proteins causes failure of cytokinesis leading to theformation of binucleated cells. Time-lapse microscopy shows that onlymidbody abscission but not further upstream events, such as furrowing,are affected. These results indicate that successful completion ofcytokinesis requires a SNARE-mediated membrane fusion event, and thatthis requirement is distinct from exocytic events that may be involvedin prior ingression of the plasma membrane.

[0008] The present invention is based, at least in part, on thediscovery that SNARE proteins are involved in cytokinesis andinterference of the SNARE proteins mechanism inhibits cytokinesisresulting in the formation of binucleated cells, and finally, apoptosisof the binucleated cells. An embodiment of the present inventioninvolves the new uses of the SNARE protein mechanism for use inscreening for anti-tumor agents. It has not been known in the art to usethe SNARE protein mechanism in a screening assay for agents for use asanti-tumor compounds.

[0009] A non-limiting embodiment of the invention involves a method ofinhibiting uncontrolled cell growth in a population of cells or a tissuecomprising contacting said cells with an effective amount of aninhibitor agent of SNARE proteins to cause formation of binucleatedcells in the population and eventual apoptosis of the cells. Such agentscan include, but are not limited to, natural products, DNA constructs,toxins, antibodies, or competing analogs of SNARE proteins.

[0010] A further non-limiting embodiment of the invention involves amethod of inducing apoptosis in tumor cells comprising contacting saidtumor cells with an effective amount of an agent that inhibits SNAREproteins and their function. Such agents can include, but are notlimited to, natural products, DNA constructs, toxins, antibodies, orcompeting analogs of SNARE proteins.

[0011] A further non-limiting embodiment of the invention involves amethod of identifying an agent that inhibits uncontrolled cell growth byinterfering with the SNARE protein mechanism comprising contacting asample of said cells with an agent to be tested that inhibits SNAREproteins and their function; monitoring the cell sample or tissue forprevention or inhibition of cytokinesis; whereby prevention orinhibition of cytokinesis in said sample is indicative of an agent thatinhibits uncontrolled cell growth.

[0012] A further non-limiting embodiment of the invention involves amethod of identifying an agent that induces apoptosis in one or moretumor cells, the method comprising contacting a sample of the cells withan agent to be tested; monitoring the cells in the sample for:prevention or inhibition of cytokinesis, formation of binucleated cells,and or cell apoptosis; whereby prevention of cytokinesis, the formationof binucleated cells, and or cell apoptosis in the sample is indicativeof an agent that induces apoptosis.

[0013] A further non-limiting embodiment of the invention involves acomposition comprising an agent that inhibits SNARE proteins inducesapoptosis in cells, and an acceptable carrier. Alternatively, thecomposition may comprise an agent that inhibits SNARE proteins andinhibits uncontrolled cell division or growth, and an acceptablecarrier.

[0014] A further non-limiting embodiment of the invention involves amethod of stimulating cell growth comprising introducing nucleic acidencoding a SNARE protein whereby said nucleic acid is expressed.Alternately, a composition comprising an agent that enhances SNAREprotein expression may be useful in stimulating cell growth.

[0015] The present invention further relates to the use SNARE proteinantibodies or to the use of anti-sense SNARE protein compounds for useas anti-tumor agents. Such agents may be used in combination withexisting and new treatment therapies, such as drugs and radiation, whichinduce apoptosis in tumor cells or which increases the sensitivity oftumor cells to these therapy modalities.

[0016] Embodiments of the present invention further comprise a method ofinhibiting tissue growth comprising: administering to cells of saidtissue an effective midbody abscission inhibiting amount of acomposition comprising a SNARE dominant negative inhibitor and apharmaceutically acceptable carrier. The tissue may be a neoplasm,sarcoma, carcinoma, neural sarcoma, leukemia, lymphoma, and combinationsof these. The method may further include a treatment such aschemotherapeutics, radiation, homeopathics, and combinations of these.

[0017] Another embodiment of the present invention is a method ofidentifying compositions that will be useful in preventing midbodycleavage in a sample of cells during cell division including determiningwhether the composition prevents midbody cleavage during cell divisionby the number binucleated cells formed in the sample; an increasednumber of binucleated cells in the sample being an indication that thecomposition prevents midbody cleavage during cell division. This methodmay further include controlling cell division in a sample of cellscomprising; administering a therapeutically effective amount of a SNAREinhibiting composition which was identified above to the sample of cellsin need thereof to inhibit midbody cleavage of said cells; thecomposition for preventing midbody clevage in cells may includeexpressing a functionally inhibiting SNARE isoform with an adenovirusvector with a tetracycline-regulatable promotor or by transienttransfection of plasmid DNA. The method may involve a sample of cells ortissue which include cells from a neoplasm.

[0018] In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

[0019]FIG. 1: Syntaxin 2 localizes to the midbody during cytokinesis.(A) Co-immuno-localization of syntaxin 2 (green) and β-tubulin (red) inNRK cells during the midbody-stage of cytokinesis. Nuclei are stainedwith DAPI (blue). (B) Competition with purified antigen eliminatessyntaxin 2-specific staining. Neither syntaxin 3 (C) nor syntaxin 4 (D)localize to the midbody of dividing cells. Scale bars, 5 μm. (E)Immunoblot analysis of NRK cell lysates shows that syntaxins 2 and 4 areabundantly expressed in NRK cells whereas the expression level ofsyntaxin 3 is relatively low. Equal amounts (15 μg protein) of total ratkidney (RK) lysates were used as controls. Molecular weight makers areindicated in kDa;

[0020]FIG. 2: Expression of soluble syntaxin 2 inhibits midbodyabscission resulting in binucleated cells. (A) Syntaxin 2D, a spliceisoform lacking a transmembrane anchor, was expressed in MDCK cells.Immuno-staining for syntaxin 2 (green) reveals expressing cells.Binucleated cells are denoted by asterisks; scale bar, 5 μm. (B)Binucleated cells formed after 16 hour expression of syntaxin 2D weresubjected to double-immunostaining for the Nuclear Transport Factor p97(green) and the tight junction protein ZO-1 (red). Note that the nucleiof binucleated cells exhibited normal p97-staining indicating thatnuclear division and reformation of the nuclear envelopes wasunaffected. (C) Frames of time-lapse phase contrast microscopy of MDCKcells expressing syntaxin 2D. For orientation, the cell if interest ishighlighted by asterisks and the midbody is circled. Quantification offailed cytokinesis after 16 hour expression of the full-length syntaxin2A or the truncated syntaxin 2D using adenovirus vectors calculated asthe fraction of nuclei in binucleated cells as the percentage of thetotal nuclei. As negative controls, syntaxin expression was prevented bythe addition of doxycycline (+DOX). The total numbers of nuclei countedfor each condition are indicated. (E) Failed cytokinesis after 24 hourexpression of syntaxin 2A, syntaxin 2D, or truncated versions ofsyntaxins 3 or 4 lacking transmembrane anchors by plasmid-mediatedtransient transfection.

[0021]FIG. 3: Endobrevin co-localizes with syntaxin 2 on the midbody.(A) Cellubrevin (green) localizes to intracellular vesicles in latetelophase NRK cells but not to the midbody which is identified byimmuno-staining for P-tubulin (red). (B) Endobrevin (green) localizes tomidbody in NRK cells. The immuno-staining for endobrevin is eliminatedby competition with total bacterial lysate containing GST-endobrevin (C)but not by lysate containing GST (D). (E) Co-immunostaining for syntaxin2 (green) and endobrevin (red) reveals their co-localization on themidbody of NRK cells. Scale bars, 5 μm;

[0022]FIG. 4: Endobrevin function in cytokinesis. (A) Expression oftruncated endobrevin lacking its transmembrane anchor (green) in MDCKcells for 16 hours results in the formation of binucleated cells(denoted by asterisks). (B) Quantification of failed cytokinesis afterexpression of truncated endobrevin or syntaxin 2A as a control for 16hours. As a further control, expression was suppressed by doxycycline(DOX);

[0023]FIG. 5: Formated alignments of (SEQ ID NO:11) Rat Syntaxin 2C and(SEQ ID NO: 13) Human Epimorphin-B;

[0024]FIG. 6: Alignment of protein sequences of four known (SEQ IDNO:9-13) Syntaxin 2 splice-isoforms in rat.

[0025]FIG. 7: Over expression of Syntaxin 2C and Syntaxin 2D in MDCKcells causes cell death;

[0026]FIG. 8: Over expression of Endobrevin in MDCK cells causes celldeath.

[0027]FIG. 9: Caspase inhibitors prevent syntaxin 2D-induced apoptosis.

DETAILED DESCRIPTION OF THE INVENTION

[0028] A highly conserved set of membrane proteins has been identifiedthat are involved in many types of intracellular fusion. These proteinslocalize to both vesicle and target membranes, and are known as solubleNSF attachment protein receptors (SNAREs); NSF stands forN-ethyl-maleimide-sensitive fusion protein. SNAREs appear to functionthroughout the secretory pathway as the minimal machinery drivingmembrane fusion. SNAREs may be conveniently divided into v-SNAREs andt-SNAREs depending upon their localization to a vesicle or a targetmembrane in a cell fusion event.

[0029] Proteins in the SNARE superfamily include SNAP-25, syntaxin (alsoknown as t-SNARE), and the vesicle-associated membrane protein (VAMP,also known as v-SNARE). These SNAREs were identified in the sea urchinegg in association with cortical granules, secretory vesicles whosecontents give rise to the fertilization envelope. Syntaxin and VAMP arealso present throughout embryogenesis enriched in cells with elevatedlevels of regulated secretion. During the cleavage stage of this embryo,a period of cell division every 45-60 min, there is enrichment of thesemolecules on vesicles accumulating at the cortex of cells, suggestingthat these vesicles may play an important role in cell division. Thus,it is likely that these proteins not only mediate the complex array ofmembrane fusion events of secretion, as previously documented, but alsofunction in the contribution of new membrane to the cell surface duringdivision.

[0030] While not wishing to be bound by theory, it appears midbodycleavage during cell division, cytokinesis is accomplished by aSNARE-mediated mechanism in mammalian cells. The SNARE-mediatedmechanism involves, but is not limited to, syntaxin 2 (also known asepimorphin) and endobrevin (also known as VAMP-8). In embodiments of thepresent invention, functional control of SNAREs have been demonstratedto regulate midbody abscission. Regulation may refer to eitherprevention or stimulation of cell growth, division, or cell apoptosis.In preferred embodiments inhibition of SNAREs results in the formationof binucleated cells. Method of identifying the location of such SNAREs,for example (SEQ ID NO: 1-13) in the midbodies of cells are disclosed asare methods of expressing and identifying compositions which inhibit thefunctionality of these proteins.

[0031] Typically, endogenous expression levels of SNAREs are often lowwhich makes detection challenging. The localization of SNAREs may bedetermined in cells by immunofluorescence microscopy including methodsfor signal amplification, antigen retrieval and suppression of antibodycross-reactivity. To define which trafficking pathway a SNARE ofinterest is involved in, one needs to specifically inhibit its function.One technique to accomplish this is to introduce inhibitors of SNAREfunction—such as antibodies or clostridial toxins—into cells by plasmamembrane permeabilization using the bacterial toxin streptolysin-O ormicroinjection. Using antibodies against syntaxin 2 and endobrevinSNAREs, the antibodies appear to localize to the region of the midbodyduring cytokinesis.

[0032] Midbody-cleavage may be prevented by interfering with thefunction of either syntaxin 2 or endobrevin in these cells.Interestingly, inhibition of syntaxin 2 or endobrevin function does notinterfere with midbody-formation, but does prevent midbody cleavage.Inhibition of midbody-cleavage eventually leads to binucleated cells andsubsequent cell death or apoptosis. Over-expression of syntaxin 2isoforms that lack a C-terminal hydrophobic domain inhibit or preventcytokinesis. Similarly, over-expression of a C-terminally truncatedversion of endobrevin inhibit or prevent cytokinesis. Thesemanipulations of syntaxin 2 lead to the formation of binucleated cellsas shown by fluorescence microscopy and by time-lapse microscopy.Binucleated cells that have formed due to failure of midbody-cleavageeventually apoptose. This involves caspases. Caspases inhibitors, suchas but not limited to ZVAD and BD may be used with functional inhibitorsof syntaxins, like syntaxin 2D, to control apoptosis in a sample ofcells as illustrated in FIG. 9.

[0033] Therefore, the methods and compositions to interfere with aSNARE-mediated mechanism and prevent cytokinesis thereby inhibiting celldivision include, but are not limited to, inhibition of SNAREs duringcytokinesis, inhibition of SNAREs such as syntaxin 2 and or endobrevinin cells during cytokinesis, and inhibition of SNAREs such as syntaxin 2and or endobrevin localized to the midbody of cells during cytokinesis.Preferably the methods and compositions of the present invention inhibitor prevent syntaxin 2 or endobrevin function through the action ofover-expressed of syntaxin 2 or endobrevin isoforms. Although methods tointerfere with a SNARE-mediated mechanism and prevent cytokinesisthereby inhibiting cell division have numerous applications, onespecific application is for use in cancer therapy to inhibit celldivision. Existing methods often have side effects on non-tumor cells.In theory, the inhibition of midbody cleavage using this techniqueshould only affect rapidly dividing cells and may be more specific thanexisting methods. Methods can be devised to express the inhibitors ofthe identified SNARE proteins in tumor cells, e.g. by viral genetransfer. This method may prove useful in the treatment of cancers.

[0034] The sequence listing for cDNA encoding human epimorphin (syntaxin2), SEQ ID NO: 1, and cDNA encoding the B isoform of human epimorphin,SEQ ID NO: 2, have been reported (Hirai et al 1994). The sequencelisting for homo sapiens vesicle-associated membrane protein 8(endobrevin) (VAMP8), mRNA, SEQ ID NO: 3, have also been reported (Wonget al 1998). The methods of the present invention may be used to preparecompositions comprising truncated non-membrane anchored or dominantnegative inhibitors of SNAREs using various vectors, or regulated vectorsystems for expressing them in both human, and rat, and other mammals.The methods of embodiments of the present invention may be used todetermine whether SNAREs such as human empimorphin (SEQ ID NO:1) orhuman endobrevin (SEQ ID NO:3) localize to the midbody of cells in asample of cells or tissue during cytokinesis. The methods of variousembodiments of the present invention may also be used to identifywhether the known or unknown truncated human isoform inhibitors of thesehuman SNAREs are effective in forming non-functional complexes withSNAREs in cells or SNAREs localized to the midbody of such cells. Suchnon-functional SNARE complexes may form binucleated cells, preventcytokinesis, and or causing cell apoptosis. Similarly, in accordancewith the methods described herein, one may identify compounds or drugformulations that mimic the effect of the truncated forms of syntaxin 2.

[0035] The present invention relates to the use of inhibitors of SNAREproteins in the prevention of cytokinesis to prevent uncontrolled cellgrowth and division. A method of preventing cell division in acollection of cells may include administering to a sample of cells ortissue an effective amount of a composition inhibiting SNARE functionduring cytokinesis in the cells. The methods and composition of thepresent invention may also be used as an assay screen for other relatedinhibitors or agents that prevent cytokinesis. This embodiment of thepresent invention relates to identification of SNAREs within cellsincluding those localized in the midbody region of dividing cells, theexpression of inhibitors to these SNAREs, and identification ofinhibitors of SNAREs in preventing cell division based on the formationof binucleated cells or prevention of midbody abscission. Such methodsand compositions may be used with cells in benign or malignant neoplasiaand may include tumors such as but not limited to sarcomas, carcinomas,lymphomas, leukemias, or neoplasms of the nervous system.

[0036] Syntaxin 2 appears to localize to the midbody. Membrane fusionevents in intracellular vesicle trafficking pathways are generallymediated by proteins of the SNARE super-family which consists of severalsub-families including syntaxins (Chen and Scheller, 2001; Jahn andSudhof, 1999; Weimbs, et al., 1997). Analysis of the localization ofsyntaxin 2 in cultured proliferating NRK (normal rat kidney) cells ledto the following serendipitous finding. An affinity-purified antibodyagainst syntaxin 2 strongly labeled small structures in a fraction ofthe cell population. Co-labeling for β-tubulin identified thesestructures as midbodies. Syntaxin 2 immunoreactivity localized todistinct regions of ˜1 μm apparent diameter on either side of themidbody (FIG. 1A). These syntaxin 2 regions were intersected bymicrotubules. This staining pattern was consistently observed using twoindependently raised syntaxin 2 antibodies and could be eliminated bycompeting antigen indicating that it is specific as illustrated in FIG.1B. Identical staining patterns were also observed with several othermammalian cell lines including (Chinese Hamster Ovary) CHO cells andhuman HEK293 cells (not shown) and illustrates that this technique maybe applied to other syntaxins and cells to identify SNARE type and theirlocation in dividing cells.

[0037] Syntaxin 2 is a ubiquitously expressed t-SNARE (Bennett et al.,1993) that has been reported to be targeted to the plasma membrane inseveral cell types including polarized Madin Darby canine kidney (MDCK)cells (Li et al., 2002; Low et al., 1996; Low et al., 2000). Two otherwidely expressed plasma membrane t-SNAREs are syntaxins 3 and 4 (Li etal., 2002; Low et al., 1996). Western blot analysis showed that NRKcells express all three syntaxins (FIG. 1E). However, neither syntaxin 3nor syntaxin 4 exhibited the same midbody localization as syntaxin 2during cytokinesis (FIG. 1C, D).

[0038] The subcellular steady-state location of a given t-SNAREsgenerally corresponds to the site at which the t-SNARE functions. Thelocalization of syntaxin 2 at the midbody therefore suggested that itmay be involved in a fusion event during cytokinesis. Syntaxin 2 may beinvolved in increasing the cell surface area during furrowing bymediating the fusion of vesicles with the plasma membrane close to thesite of ingression. Syntaxin 2 may be directly involved in the finalabscission of the midbody to result in completely separated daughtercells.

[0039] Syntaxin 2 function during cytokinesis. To investigate whatfunction syntaxin 2 plays in cytokinesis and to examine its mechanism ofaction a dominant-negative approach was employed. The over-all domainstructure of syntaxins is highly conserved (Weimbs, et al., 1997;Weimbs, et al., 1998), and they are characterized by a C-terminaltransmembrane anchor while the rest of the molecule protrudes into thecytoplasm. Recombinant soluble SNAREs that lack their membrane anchorsare known to inhibit membrane fusion by forming nonfunctional complexeswith endogenous SNARE proteins (Hua and Scheller, 2001). Abrain-specific, alternatively spliced isoform of syntaxin 2, termedsyntaxin 2D, has previously been identified that lacks a transmembraneanchor, it is a truncated mutant, while the remainder of the cytoplasmicdomain is identical to full-length syntaxin 2 (Quinones et al., 1999).The function of syntaxin 2D is unknown. However, it was reported to be asoluble cytoplasmic protein (Quinones et al., 1999) and would bepredicted to act as a dominant-negative inhibitor of the function ofmembrane-anchored syntaxin 2.

[0040] In an embodiment of the present invention, syntaxin 2D, rat, wasexpressed in MDCK cells using an adenovirus vector with atetracycline-regulatable promotor. Other virus vectors and regulatorsmay be used as would be obvious to try by those skilled in the art.Immunofluorescence analysis confirmed the cytoplasmic localization ofsyntaxin 2D (FIG. 2A). Syntaxin 2D expression for 16 hours resulted in ahigh frequency of binucleated cells indicating that the cells hadundergone nuclear division in the absence of cytokinesis (FIG. 2A, B).Similarly, it was observed that overexpression of syntaxin 2C resultedin an increase in the fraction of binucleated cells formed in MDCKcells. This effect could be prevented by suppressing syntaxin 2C or 2D,FIG. 2 and FIG. 7, expression by the addition of doxycycline indicatingthat the observed block of cytokinesis is not due to the adenoviralinfection. Furthermore, adenovirus-mediated expression of themembrane-anchored, full-length syntaxin 2A did not result in an increasein binucleated cells (FIG. 2B). These results indicated that syntaxin 2function is involved in cytokinesis and that composition inhibitingSNARE function during cytokinesis or mid-body abscission will causebinucleated cells to form. Such compositions inhibiting SNARE functionmay be used to control or prevent cytokinesis or midbody abscission.Given that only ˜50% of the cells underwent mitosis during the course ofthese experiments it is estimated that cytokinesis failed inapproximately 60% of the mitotic events in cells that expressed syntaxin2D. The expression of this isoform may form the basis of a method ofpreventing mid-body cleavage in cells which comprises administering tocells an effective amount of a composition inhibiting mid-body cleavageof the cells. Further, the immunofluorescence and counting of cellsserve as an assay for determining whether any expressed isoform isfunctional for inhibiting midbody cell abscission.

[0041] As a further control for the specificity of the dominant-negativeinhibition of syntaxin 2 function, truncated versions of syntaxins 3 and4—lacking the transmembrane anchors—were expressed in MDCK cells bytransient transfection of plasmid vectors. This was compared totransient transfection of syntaxin 2A or 2D cDNAs inserted intoidentical plasmid vectors. Similar to the adenoviral gene transferabove, expression of syntaxin 2D for 24 hours resulted in a highfrequency of binucleated cells (FIG. 2E). In contrast, neitherexpression of the membrane-anchored syntaxin 2A nor of the truncatedsyntaxins 3 or 4 (human) had this effect. This result indicates that thedominant-negative inhibition by non-membrane anchored syntaxins isspecific, and that syntaxin 2 is specifically involved in cytokinesis.

[0042] Syntaxin 2 function during midbody abscission. To distinguishwhether syntaxin 2 inhibition prevents the ingression of the cleavagefurrow or the abscission of the midbody, syntaxin 2D-expressing cellswere investigated by time-lapse microscopy. FIG. 2C shows representativeframes. In 6 independent time-lapse experiments, 41 events were observedthat resulted in the formation of binucleated cells. In all cases,nuclear division, cleavage furrow formation and ingression, and theformation of midbodies were indistinguishable from controls. However,the cells were unable to undergo midbody abscission. The average timethat the syntaxin 2D-expressing cells remained in the midbody stage was153 min (range 64-355 min, n=41) after which midbody regression occurredto lead to binucleated cells. Syntaxin 2D or cells treated to expresssyntaxin 2D may be used to form a composition comprising which inhibitsmid-body abscission in cells. This method may be used to identifycompositions that will inhibit/prevent midbody cleavage during celldivision and comprises determining whether the composition inhibits orprevent midbody cleavage during cell division by the number binucleatedcells formed in the treated sample; an increased number of binucleatedcells being an indication that the composition inhibits or preventsmidbody cleavage during cell division. The result of this test may beused to identify compositions that prevent midbody cell abscission in acell population, and such compositions may then be administered in atherapeutically effective amount to cells in need of prevention orinhibition of midbody abscission.

[0043] To investigate whether syntaxin 2 inhibition might affect thereassembly of the nuclear envelope, the binucleated cells wereimmuno-stained with antibodies against the Nuclear Transport Factor p97(FIG. 2D) or lamin B2 (not shown). The nuclear envelopes of thebinucleated cells appeared to be complete and intact and wereindistinguishable from those of control cells indicating that syntaxin2-inhibition has no effect on the nuclear envelope and that nucleardivision had occurred unperturbed. Evidence for micronuclei or nuclearbuds in the binucleated cells after syntaxin 2-inhibition was notobserved. These defects would be indicators of loss or malsegregation ofchromosomes as a result of defects in the spindle, centromeres or as aconsequence of chromosome undercondensation (Fenech and Crott, 2002).Overall, these results indicate that syntaxin 2 functions duringmidbody-abscission but its inhibition does not appear to affect furtherupstream events of mitosis such as chromosome segregation, nuclearenvelope reassembly, furrowing etc. Furthermore, the results indicatethat midbody-abscission involves a SNARE-mediated fusion event, andsuggest that this event requires a different fusion machinery thanexocytosis for the delivery of new membrane to aid in furrow ingression.Finally, if midbody abscission is blocked by syntaxin 2-inhibition,cells can not otherwise “rip apart” or “pinch off” to completecytokinesis.

[0044] Endobrevin/VAMP-8 functions together with syntaxin 2 duringmidbody abscission. If syntaxin 2 mediates a membrane fusion event thatsevers the midbody, it would be predicted to involve other members ofthe SNARE machinery as well. In other intracellular fusion events, smallv-SNAREs of the synaptobrevin/VAMP family mediate membrane fusion inconcert with syntaxins. A v-SNARE involved in cytokinesis would beexpected to exhibit a relatively ubiquitous tissue expression pattern.Experiments were performed to investigate whether the two ubiquitouslyexpressed v-SNAREs cellubrevin/VAMP-3 or endobrevin/VAMP-8 localize tothe midbody region during cytokinesis. FIG. 3A shows that cellubrevin isonly found on intracellular vesicles but not at the midbody duringcytokinesis. In contrast, endobrevin appears to be highly concentratedat the midbody in a staining pattern very similar to that of syntaxin 2(FIG. 3B, D). Again, the immuno-signal could be eliminated bycompetition with antigen (FIG. 3C), and two independent endobrevinantibodies resulted in identical staining patterns (not shown).Double-immunofluorescence microscopy with antibodies against syntaxin 2and endobrevin revealed nearly completely overlapping localizations(FIG. 3E).

[0045] To investigate whether endobrevin is functionally involved incytokinesis, an isoform or truncated mutant of endobrevin, human,lacking the C-terminal transmembrane anchor was prepared and wasexpressed using a tetracycline-regulatable adenoviral vector asdescribed above for syntaxin 2D. FIG. 4A shows that expressed truncatedendobrevin distributes throughout the cytoplasm and results in a highpercentage of binucleated cells after 16 hours. As a control, when theexpression of truncated endobrevin was prevented by the inclusion ofdoxycycline, the formation of binucleated cells was suppressed (FIG.4B). These results indicate that truncated endobrevin acts as adominant-negative inhibitor of membrane-anchored endobrevin resulting ininhibition of cytokinesis. These results indicated that endobrevinfunction is involved in cytokinesis and that composition inhibitingSNARE function during cytokinesis or mid-body abscission will causebinucleated cells to form. Such compositions inhibiting SNARE functionmay be used to control or prevent cytokinesis or midbody abscission.Time-lapse microscopy revealed that endobrevin inhibition did notinterfere with events upstream of midbody-formation but resulted in theinability to cleave the midbodies. The average time betweenmidbody-formation and regression into binucleated cells was 173 min(range 80-345 minutes, n=45). This phenotype was indistinguishable fromthat of syntaxin 2-inhibition as described above, suggesting thatsyntaxin 2 and endobrevin may act together at the same step duringmidbody abscission.

[0046] Collectively, these results show for the first time that midbodyabscission involves the action of members of the SNARE membrane fusionmachinery. Inhibition of syntaxin 2 or endobrevin had no apparent effecton cleavage furrow invagination, nuclear division, reformation of thenuclear envelope or other mitotic events. It is therefore unlikely thatthese SNAREs are involved in any step prior to midbody abscission. Sincecleavage furrow invagination is believed to require exocytosis for theinsertion of additional plasma membrane it is likely that other SNAREsare involved in this process in mammalian cells. A possible candidate issyntaxin 4 which has been found to localize to the ingressed plasmamembranes separating the prospective daughter cells prior to midbodyabscission (see FIG. 1D). This would be analogous to the proposedfunction of Syn-4 in C. elegans (Jantsch-Plunger and Glotzer, 1999).Note that mammalian and C. elegans SNAREs are too divergent to allowassignment of orthologues by sequence comparison (Jantsch-Plunger andGlotzer, 1999; Weimbs, et al., 1997). Therefore, despite thecoincidental similarity of their names, it remains to be establishedwhether the mammalian syntaxin 4 may have the equivalent role of the C.elegans Syn-4 in cleavage furrow ingression.

[0047] Results of embodiments of this invention show that syntaxin 2 andendobrevin appear to function in midbody abscission during celldivision. Further, embodiments of the present invention provide methodsfor identifying the location of SNAREs in cells undergoing cytokinesis,methods for making and delivering isoforms composition of such SNAREs,and methods for identifying whether cells treated with such SNAREisoforms result in the formation of binucleated cell. The methods andcompositions of the present invention may be applied to mammalian cellsincluding human cells for which SNARE proteins and DNA for theseproteins are known (SEQ ID NO: 1-3, SEQ ID NO: 13). The terminal step ofcytokinesis utilizes a SNARE machinery or function that is distinct fromthose involved in prior steps that require membrane fusion such asfurrowing. If the function of syntaxin 2 or endobrevin is inhibited bySNARE isoforms or other proteins which inhibit their normal function,cell division can not be completed indicating that other SNAREs can notsubstitute their function. This may mean that midbody abscission is ahighly regulated, active process, and that mammalian cells may possessno alternative mechanisms that can accomplish the breakage of thisnarrow bridge. These results may be used in a method of preventingmid-body cleavage in cells comprising administering to cells aneffective amount of a composition inhibiting the function of such SNAREsin mid-body cleavage or abscission of cells.

[0048] Cell division is not only a fundamental biological process but isalso of particular interest as a target for anti-tumor strategies.Currently used anti-tumor compounds target the cell cycle at varioussteps. The identification of molecules involved in the terminal step ofcytokinesis may provide potential new targets that may be exploited forcancer therapy.

[0049] Affinity-purified antibodies against the cytoplasmic domains ofrat syntaxins 2, 3 and 4 have been described previously (Low et al.,2000). An antibody against the cytoplasmic, domain of endobrevin wasraised and affinity-purified equivalently as described previously (Li etal., 2002). As confirmatory controls, independently raisedaffinity-purified antibodies against syntaxin 2 (Quinones et al., 1999)and endobrevin (gift from Wanjin Hong, IMCB, Singapore) were used. Amonoclonal β-tubulin antibody developed by Michael Klymkowsky wasobtained from the Developmental Studies Hybridoma Bank, The Universityof Iowa. Antibodies against the Nuclear Transport Factor p97 and ZO-1were from ABR (Golden, Colo.) and Chemicon (Temecula, Calif.),respectively.

[0050] Cell culture and immuno-localization; NRK cells (from ATCC) werecultured in DMEM with sodium pyruvate, 10% FBS and penicillin andstreptomycin. MDCK cells were cultured as described (Low et al., 2000).Cells were fixed in methanol and subjected to immuno-staining andconfocal fluorescence microscopy as described previously (Low et al.,2000). For localizing simultaneously two proteins recognized by rabbitprimary antibodies (syntaxin 2 and endobrevin), fluorescein-labeled Fabfragments of the secondary antibody (Jackson ImmunoResearch, West Grove,Pa.) were used after incubation with the first rabbit primary antibody.The cells were briefly fixed again with 4% paraformaldehyde, thenincubated with the second rabbit primary antibody, followed by TexasRed-labeled secondary antibody (Weimbs, et al., 2003). Antibodyconcentrations were titered so that all negative controls were negative.

[0051] Expression of SNARE cytoplasmic domains; the adenovirus vectorsfor tetracycline-regulated expression of rat syntaxins 2A and 2D havebeen described previously (Quinones et al., 1999). The identical vectorsystem was used for the expression of truncated endobrevin lacking itstransmembrane domain. MDCK cells stably expressing theTET-transactivator (Clontech, Palo Alto, Calif.) were infected withvirus numbers titered to result in 90% of expressing cells after 16hours. After fixation, double-immuno-staining for the respectivetruncated SNARE and the 6.23.3 endogenous plasma membrane marker (Low etal., 2000), and nuclear staining with DAPI, random fields were imaged byfluorescence microscopy, and the number of mono- and bi-nucleated cellswere counted manually.

[0052] Truncated SNAREs were expressed in MDCK cells as described above.˜8 hours post-infection, cells were subjected to time-lapse phasecontrast microscopy (2 minutes/frame) using a fully motorized LeicaDMIRB microscope equipped with a temperature-, CO₂— andhumidity-controlled environmental chamber. Images were processed usingMetamorph, Adobe Photoshop and Quicktime.

[0053] Time-lapse phase contrast microscopy of MDCK cells, FIG. 2C,expressing syntaxin 2D were recorded at 2 minutes/frame and a movierecorded at 10 frames/second. The experiment shows cell with failingcytokinesis due to expression of syntaxin 2D.

[0054] Time-lapse phase contrast microscopy of MDCK cells expressingtruncated endobrevin were recorded at 2 minutes/frame and a movierecorded at 10 frames/second. The experiment shows cells with failingcytokinesis due to expression of truncated endobrevin.

[0055] The endogenous expression levels or most SNAREs are relativelylow (with the exception of neuronal SNAREs in neurons) which can makedetection a challenge. Below are descriptions of methods for enhancingsignals in immunofluorescence experiments using cultured cells or tissuesections. Protein and nucleotide sequences for human and rat syntaxins,endobrevin, and their isoforms are given in Table 1 and in the sequencelisting. TABLE 1 SEQ ID NO DESCRIPTION 1 Human epimorphin cDNA 2 IsoformB of human epimorphin cDNA 3 Homo sapiens membrane protein 8(endobrevin) mRNA 4 Rattus norvegicus endobrevein mRNA 5 Rattusnorvegicus Syntaxin 2A, Syntaxin 2, mRNA 6 Rattus norvegicus Syntaxin2B, Syntaxin 2′, mRNA 7 Rattus norvegicus Syntaxin 2C, Syntaxin 2″, mRNA8 Human epimorphin, mRNA 9 Rat syntaxin 2A, Syntaxin 2 protein 10 Ratsyntaxin 2B, Syntaxin 2′ protein 11 Rat syntaxin 2C, Syntaxin 2″ protein12 Rat syntaxin 2D protein 13 Human epimorphin B protein

[0056] Immunofluorescence-staining with signal amplification byanti-fluorescein tertiary antibody. This method uses a primary antibodyagainst a SNARE protein, followed by a fluorescein-labeled donkeyanti-rabbit-IgG secondary antibody. To enhance the signals, a rabbitantibody is then used that recognizes the fluorescein and is coupled tothe fluorophore Alexa-488 (Molecular Probes Eugene, Oreg., cat#A-11090). Since the spectral properties of Alexa-488 are nearlyidentical to fluorescein, the result is an amplification of the “FITC”signal.increases of approximately five-fold. The method comprises:

[0057] (1) MDCK cells are cultured on 12 mm Transwell filters in MEMcontaining 10% FBS. The cells are allowed to polarize for at least 3days, with changes of media every other day; (2) Rinsed cells briefly 3times with PBS containing 1 mM each CaCl2 and MgCl2 (PBS+); (3) Fix with4% paraformaldehyde in PBS+ for 20 min at room temperature. Duringfixation, the cells are placed on an orbital shaker at very low speed(alternatively, cells can be fixed in cold methanol at 20° C. for 10min); (4) Quench any remaining fixative with 75 nM NH4Cl and 20 mMglycine (both from a 1 M stock) in PBS at RT for 10 min with shaking (incase of the methanol fixation, the quenching step is omitted); (5) After2 brief rinses in PBS, the cells are blocked and permeabilized in BlockSolution (PBS containing 3% BSA and 0.2% TX-100) at 37° C. for 30 min;(6) Primary antibody diluted in Block Solution is centrifuged at 12,000g for 15 min to pellet any aggregates; (7) The membrane is carefully cutout of the Transwell mount and placed on parafilm on a 30 μl drop of theantibody. Another 30 μl of the diluted antibody is placed on tope of thefilter. Incubation is in a humid chamber at 37° C. for 2 hours; (8) themembrane is transferred back to a 12 well dish and washed 4 times 5 minin Wash Solution) PBS with 0.05% TX-100 and 0.7% fish skin gelatin(Sigma Cat # G-7765, St. Louis, Mo.); (9) the secondary antibodyconjugated with fluorescein is diluted in Wash Solution and againcentrifuged for 15 min to pellet out any aggregates and applied to themembrane as described for the primary antibody. Incubation is for 1 hrat 37° C. in a humid chamber; (10) the membrane is washed 4 times 5 minin Wash Solution and the Alexa 488 conjugated anti-fluorescein isdiluted in Wash Solution and applied to the membrane aftercentrifugation as described previously. The antibody is allowed toincubate for 1 hr at 37° C.; (11) excess antibody is removed by washing4 times 5 min in Wash Solution and 2 times 3 min in PBS containing 0.1%TX-100, followed by 2 rinses in PBS; and (12) the membrane is mountedcell side up and is ready for viewing under the microscope.

[0058] Antigen Retrieval by pressure-cooking. Immuno-fluorescencestaining of SNAREs in sections of fixed tissues often results in weaksignals. This may be due to the fact that SNAREs appear to spend most oftheir time in complexes with other SNAREs or regulatory proteins.Fixation with protein-cross-linking fixatives, like formaldehyde, maythen mask many epitopes. This could theoretically lead to localizationartifacts because a sub-population of a SNARE may be “invisible” byimmuno-staining. Several methods are used to unmask epitopes in tissuesections, including digestion with proteolytic enzymes, denaturationwith urea, SDS, or guanidine hydrochloride, and heat-treatment.Preferably heat-treatment using a pressure cooker leads to the mostreproducible signal enhancement while preserving tissue morphology.Paraffin sections of tissues from animals perfusion-fixed with 4%paraformaldehyde in PBS+ have worked well. The method comprises:

[0059] (1) Deparaffinize tissue and re-hydrate sections on slides asusual (leave slides wet until ready for pressure-cooking); (2) make 2liters of 10 mM Na-citrate buffer, pH 6.0 by dilution from 1 M stock;(3) put citrate buffer in large stainless-steel householdpressure-cooker (must not be aluminum as it reacts with the citrate).With the lid only loosely on the cooker heat until boiling; (4) placeslides in a glass or steel slide-holder and put into boiling citratebuffer; (5) close lid tightly. Place the weight on the pressure-cooker'svalve; (6) continue to heat until weight starts to wobble; (7) heat forone more minute; (8) remove cooker from heater, place under running coldwater tap; (9) once pressure is down, open lid and flood cooker withrunning cold tap water; (10) take out slides and proceed withimmuno-staining as usual.

[0060] Suppression of antibody cross-reactivity. Immuno-localizationexperiments of endogenous proteins suffer from the inherent problem thatusually no negative control is available (in contrast to experimentswith transfected cells in which the non-transfected cells can serve asthe negative control). Omitting the primary antibody only controls forautofluorescence, background by the secondary antibody etc., but it doesnot establish specificity of the primary antibody. Competition with theantigen is better but does still not exclude cross-reactivity of theprimary antibody. Polyclonal antibodies have to be affinity-purifiedagainst the antigen. Unpurified antisera almost always lead toartifactual staining results.

[0061] . For plasmid-mediated transient transfection experiments, thecDNAs encoding syntaxin 2A or 2D or truncated versions of syntaxin 3 or4 were inserted into the vector pcDNA4/TO and transfected into MDCKcells cultured on glass cover slips using the ExGen 500 transfectionreagent (Fermentas, Inc., Hanover, Md.). After 24 hours, analysis offailed cytokinesis was carried out as described above.

[0062] Many SNAREs are closely related to each other, especially in the“SNARE” motifs (Weimbs, et al., 1997) which can lead to problems ofantibody cross-reactivity. For example, polyclonal antibodies raisedagainst GST-fusion proteins of any of the mammalian plasma membranet-SNAREs syntaxin 2, 3 and 4 cross-react slightly with the other twosyntaxins even after affinity-purification. A simple method to overcomethis problem is competitive inhibition using lysates of bacteria thatexpress the related syntaxin-GST fusion proteins (e.g. immuno-stainingfor syntaxin 2 would be carried out in the presence of syntaxin 3 and4-lysates). Because the non-specific antibodies may be against bothnative and denatured antigen, a mixture of denatured and non-denaturedlysates is added to the antibody solution before staining. The methodcomprises the following steps and compositions:

[0063] (1) Grow E. coli expressing GST-syntaxins under the appropriateconditions. Prepare a total cell lysate using a standard lysozymeprotocol. Bacteria from a 1 liter culture will lead to approximately 25ml of lysate; (2) add 250 μl SDS lysis buffer (0.5% SDS, 100 mM NaCl, 50mM trithanolamine-Cl, 100 mM NaCl, 5 mM EDTA); (3) boil the abovesolution for 10 min.; (4) Add 250 μl Triton-dilution buffer (2.5% TritonX-100 500 mM trithanolamine-Cl, 100 mM NaCl, 5 mM EDTA); (5) Mix abovesolution, add 250 μl of non-denatured bacterial lysate and store at −80°C. in aliquots; and (6) add the above mixture at 4% to the primaryanti-syntaxin antibody dilution during immunofluorescence staining.

[0064] A major strategy for defining the function of SNAREs is tospecifically inhibit the function of an individual SNARE protein andmeasure the effects on the kinetics or fidelity of membrane traffickingpathways, the targeting of cargo proteins, or parameters of epithelialcell polarity. The difficulty lies in the fact that no ideal and simplemethod is available to inhibit SNAREs efficiently and specifically.Nature has provided clostridial neurotoxins—tetanus and botulinumtoxins—which are highly specific metalloproteases that cleave andinactivate several SNARE proteins (Schiavo et al., 2000). However, mostof these toxins cleave only neuronal SNAREs such as syntaxin 1, SNAP-25and synaptobrevin which are not normally expressed in epithelial cellsTo make matters worse, clostridial neurotoxins can attach to and enterneurons but not non-neuronal cells. It is therefore necessary tointroduce these toxins by other means. The same is the case for otherinhibitory reagents such as antibodies and recombinant fragments ofSNAREs. Two methods—using permeabilized cells or microinjection—can beused for introducing these non-membrane-permeable inhibitors intoepithelial cells. An alternative strategy is to expressdominant-negative inhibitors by gene transfer.

[0065] Dominant-negative inhibition by overexpression of SNAREs. It hasbeen observed in several systems that the overexpression of a wild-typesyntaxin causes inhibition of the trafficking pathway that the syntaxinis normally involved in. Examples are syntaxin 3 in MDCK cells (Low etal., 1998), syntaxin 5 in BHK-21 cells (Dascher and Balch, 1996), andsyntaxin 4 in mast cells (Paumet et al., 2000). Although not wishing tobe bound by theory, a plausible hypothesis of the observed inhibition isthat the over-expression of one SNARE results in a stoichiometricimbalance with the other SNAREs involved in the same pathway. This maylead to the formation of non-productive, incomplete SNARE complexes andmay cause one of the other functional SNAREs (or a regulatory factor) tobecome limiting. In any case, the effect appears to be quite specific asother trafficking pathways generally remain unaffected. However,successful inhibition requires relatively high levels of overexpression.For example, overexpression of syntaxin 3 (−10×over endogenous levels)by stable transfection in MDCK cells resulted in partial inhibition ofbiosynthetic trafficking to the apical membrane, as well as apicalrecycling, however, similar overexpression of syntaxin 4 had nomeasurable effect on any pathway (Low et al., 1998).

[0066] For this reason, an expression system should be chosen thatresults in high-level expression but can ideally also be regulated.Dasher and Balch used a vaccinia virus system which allows constitutivehigh-level expression (Dascher and Balch 1996). However, it is onlyuseful for relatively short-term expression and may therefore beunsuitable to investigate long-term parameters such as development ofepithelial cell polarity. The same is the case with the usual transienttransfection approaches. A promising alternative is expression by stabletransfection using a regulatable system such as those using thetetracycline repressor or transactivator. Also useful are adenoviralvectors that express the gene of interest under tetracycline control.The ability to regulate expression of the SNARE may be important becauseinhibition of any trafficking pathway may be potentially toxic.

[0067] In all cases it is important to verify that the over-expressedSNARE is still correctly targeted. Mistargeting of the SNARE of interestmay compromise the specificity of the desired inhibition. Since toostrong overexpression of any protein may result in its mis-localization,it is again desirable to be able to regulate the expression level.Accordingly, the methods of the present invention may include the stepof verifying the over-expression of a particular SNARE (e.g. Syntoxin2D) and the inhibition of other SNAREs (e.g. Syntoxin 2B)

[0068] Dominant-negative inhibition can also be achieved by expressionof truncated SNAREs. There have been no systematic studies aiming atidentifying domains of SNAREs that may be most potent and/or specificinhibitors. But is it generally believed that a non-membrane anchoredtruncation mutant will form complexes with its cognate SNAREs that arenon-productive due to the lack of proper membrane attachment. Apotential caveat is that non-membrane anchored SNAREs generally localizethroughout the cytoplasm. Since SNARE-SNARE interactions—at least invitro—are relatively promiscuous (Yang et al., 1999), the potentialexists that a non-membrane anchored SNARE may be a less specificinhibitor than the same full-length SNARE when over-expressed.

[0069] Successful examples are the expression of the cytoplasmic domainof syntasin 4 in adipocytes (inhibits GLUT4 translocation (Olson et al.,1997) and the expression of the cytoplasmic domain of syntaxin 5 inBHK-21 cells (inhibits ER to Golgi transport (Dascher and Blach, 1996)).In both cases, vaccinia virus expression systems were used forrelatively short-term experiments (−3-6 hours post infection).Expression of non-membrane anchored mutants of syntaxin 2 andendobrevin/VAMP-8 using a replication-deficient,tetracycline-regulatable adenovirus system very effectively inhibitedthe function of the respective SNAREs in MDCK cells. In this case, thecells could be monitored for periods up to 24 hours post infection. Asan important control, the observed inhibitory effects could beeliminated by tetracycline-suppression.

[0070] These and still further objects as shall hereinafter appear arereadily fulfilled by the present invention in an unexpected manner aswill be readily discerned from the following detailed description of thepreferred embodiments of the invention, especially when read inconjunction with the accompanying drawings.

[0071] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and anyconstructs, viruses, antibodies, toxins, or proteins which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

1 12 1 867 DNA Homo sapiens 1 atgcgggacc ggctgccaga cctgacggcgtgtaggaaga atgatgatgg agacacagtt 60 gttgtggttg agaaagatca tttcatggatgatttcttcc atcaggtgga ggagattaga 120 aacagtattg ataaaataac tcaatatgttgaagaagtaa agaaaaacca cagcatcatt 180 ctttctgcac caaacccgga aggaaaaataaaagaagagc ttgaagatct gaacaaagaa 240 atcaagaaaa ctgcgaataa aattcgagccaagttaaagg ctattgaaca aagttttgat 300 caggatgaga gtgggaaccg gacttcagtggatcttcgga tacgaagaac ccagcattcg 360 gtgctgtctc ggaagtttgt ggaagccatggcggagtaca atgaggcaca gactctgttt 420 cgggagcgga gcaaaggccg catccagcgccagctggaga taactgggag aaccaccaca 480 gacgacgagc tagaagagat gctggagagcgggaagccat ccatcttcac ttccgacatt 540 atatcagatt cacaaattac tagacaagctctcaatgaaa tcgagtcacg tcacaaggac 600 atcatgaagc tggagaccag catccgagagttgcatgaga tgttcatgga catggctatg 660 tttgtggaga ctcagggtga aatgatcaacaacatagaaa gaaatgttat gaatgccaca 720 gactatgtag aacacgctaa agaagaaacaaaaaaagcta tcaaatatca gagcaaggca 780 agaaggaaaa agtggataat tattgctgtgtcagtggttc tggttgtcat aatcgttcta 840 attattggct tgtcagttgg caaatga 867 2834 DNA Homo sapiens 2 atgcgggacc ggctgccaga cctgacggcg tgtaggaagaatgatgatgg agacacagtt 60 gttgtggttg agaaagatca tttcatggat gatttcttccatcaggtgga ggagattaga 120 aacagtattg ataaaataac tcaatatgtt gaagaagtaaagaaaaacca cagcatcatt 180 ctttctgcac caaacccgga aggaaaaata aaagaagagcttgaagatct gaacaaagaa 240 atcaagaaaa ctgcgaataa aattcgagcc aagttaaaggctattgaaca aagttttgat 300 caggatgaga gtgggaaccg gacttcagtg gatcttcggatacgaagaac ccagcattcg 360 gtgctgtctc ggaagtttgt ggaagccatg gcggagtacaatgaggcaca gactctgttt 420 cgggagcgga gcaaaggccg catccagcgc cagctggagataactgggag aaccaccaca 480 gacgacgagc tagaagagat gctggagagc gggaagccatccatcttcac ttccgacatt 540 atatcagatt cacaaattac tagacaagct ctcaatgaaatcgagtcacg tcacaaggac 600 atcatgaagc tggagaccag catccgagag ttgcatgagatgttcatgga catggctatg 660 tttgtggaga ctcagggtga aatgatcaac aacatagaaagaaatgttat gaatgccaca 720 gactatgtag aacacgctaa agaagaaaca aaaaaagctatcaaatatca gagcaaggca 780 agaaggcaac aacattgtca tagcaaccat atcccaagagccatttatcc ttga 834 3 702 DNA Homo sapiens 3 aggaagccga ctaggcgaattcacttactg accggcctgg gctgctctga gacatggagg 60 aagccagtga aggtggaggaaatgatcgtg tgcggaacct gcaaagtgag gtggagggag 120 ttaagaatat tatgacccagaatgtggagc ggatcctggc ccggggggaa aacttggaac 180 atctccgcaa caagacagaggatctggaag ccacatctga gcacttcaag acgacatcgc 240 agaaggtggc tcggaaattctggtggaaga acgtgaagat gattgtcctt atctgcgtga 300 ttgtttttat catcatcctcttcattgtgc tctttgccac tggtgccttc tcttaagtaa 360 cagggaacct ctcccacctgcccttctttt cagggacaac cctccataaa tgtgtgccaa 420 gagggtctcc tttcctgtcttcctctacag agaatgctgc tcggtcctcc tacccctctt 480 cccgaggccc tgctgccacgttgtatgccc cagaaggtac cttggtcccc cggaaggaga 540 gaaaaaagag agatggactgtggctgcatt tcttgggtcc ttagagtggg ctggagagac 600 ctagagggcc cagcatgtggctgggaaact gttggtggcc agtgggtaat aaagaccttt 660 cagtatccct aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aa 702 4 303 DNA Rattus norvegicus 4 atggaggccagtgggagtgc cggaaatgac cgagtcagga acctgcagag tgaggtggag 60 ggagtcaagaatattatgac ccagaatgtg gagcggatct tggcccgagg ggagaacctg 120 gaccatctccgaaacaagac agaggacttg gaagccacgt ctgaacactt caagacgacg 180 tcccagaaggtggcccggaa gttctggtgg aagaatgtga agatgattgt catcatctgt 240 gtgattgtccttatcattct catcctcatc atactctttg ctacgggcac catccccact 300 taa 303 53003 DNA Rattus norvegicus 5 atcgccaccg cagcctaggc agagccagtc ggcccaggcccctgtctctg cctggcctca 60 gctccccgcc ggcccgccgc gcaccttacc cgcacatcccttggaggtct agccgggtgc 120 ccccagaccc cggcctccag cacagaggca agaggcagagagcagcagcg agggcgagga 180 cgaagaaggg gaggaggagc tcgtcgcagg atgaaggatcggactcagga gctgcggagt 240 gcaaaagaca gtgacgatga agaggaagtg gttcatgtggatcgagacca ctttatggat 300 gagttctttg agcaggtgga agagatccga ggctgcatcgagaaactgtc cgaggatgtg 360 gagcaagtga agaaacagca cagtgccatt cttgctgcccccaaccccga tgagaagact 420 aaacaggagc tggaggacct cacggcagac atcaaaaagacggcaaacaa ggtccggtcc 480 aagttgaaag cgatcgagca gagcattgag caggaagaggggttgaatcg ttcttctgca 540 gacctgcgta tccgtaagac ccagcactcc acactctcacggaagttcgt ggaggtaatg 600 accgaatata atgcaactca gtctaagtac cgggaccgctgcaaggaccg tatccagagg 660 cagctggaga tcactggcag gactactacc aacgaagagctggaagacat gttggaaagc 720 gggaagctgg ccatcttcac ggacgacatc aaaatggactcgcagatgac aaagcaagcc 780 ctgaatgaga tagagacaag gcacaatgag atcatcaaactggaaaccag catccgagag 840 ctgcacgaca tgtttgtgga catggccatg ctcgtggagagccagggtga gatgatcgac 900 cgaattgagt acaatgtgga acattctgtg gactacgtggagcgagccgt gtccgacacc 960 aagaaagctg tgaaatatca gagcaaggcc aggaggaagaaaattatgat catcatttgc 1020 tgtgtggtgc tgggggtggt cttggcgtca tctattggggggacactggg cttgtaggcc 1080 cctacccttc tcttccccag gaccctcccc acacatcgggagcaataccc ccaccaccct 1140 ttcactcttt cccctgctcc aagctcactc ccaaaacagacccaggcagt tccagcctct 1200 ctcaccctca cgcagaccct ggagtccctg gctctcaccttgccatggat ccccctccac 1260 cttgccgcac atagatagca gcaggcgtga tcacacatgcacaccaacat gcatgccgag 1320 ggcacatgct caagacgtgt ggacacccca gcgtgtgtgtacttgtgtag atgtatgtag 1380 atgccctgaa cctcttcttg ctgccacctt catcctgtgtggtctgaact tccctctcta 1440 gccggttctg tgctgactgt agcagcctac catggcccaacctgttctgt gtgaatagac 1500 atggtgtgta tgtgtgtgtg tgtgtgtgtg tgtgtgtgtgtgtgtgtgtg tgtgtgtgta 1560 gatctgtgcc catacaatgt atgtacatgg agttttgtgaatgtgaaaac ccaaagcatt 1620 tcctccacct tctctcctat ctgttacatt tggagtgggaggcatatgtg aggataattt 1680 acattctcag aggatccagg gtggcccggt gaccatgccacacatctttt gtgatgtaga 1740 acaaactttc ctggcttggt tactgccagg ggtcacatcatgtcctgagg attctctctc 1800 tcagcctctc tctctgtctc tctgtccctc agtctctgtctctgtctctg tctctgtgtg 1860 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtc cacatgtatgtaacccctag attagctgtg 1920 ctgaggaagg aggctgcctg tccattccag aatattctgctgcagggccc agcacctcct 1980 gcccccacct tggcttcttt ggagatgtca gaggaggccccaggatctag ttcccttctt 2040 ctttcagccc agccagaact cccattaact cagatgtttctagcagcctc cctgacaccc 2100 cagtcatggt gttcacaagg acagggctgt ggctggcatgctgctcctgc ctctttgaat 2160 ggcactgggc ccacagctgc ccaccactga gcctcaaacagatgagccgt ccctgagagt 2220 ctgctgtaac ccagctttct aagacccaca cctcacacgatggacatgtt cgttgactgc 2280 tgtccacatg tgtatcctgg atggttgtct gggggctggtgtctgttgca tgttctcaga 2340 tgtgtgctgc gtgccctcca cacaccccaa accatcaagcccaattatac tcccttggtg 2400 gtcccaggcc caagccagga ctcaatgctt ccattctccctttccttgcc tcttacaaag 2460 cacctgcgtg tccatctcat gtgcccatgg gtcatcatgctcccgtcata ccttgagcgt 2520 gtacacatgt gtgttctatg cactcgccct gccctacctacccaacagag gacaagatgg 2580 tcggccccta gctcccttct cccccaccta gcctttccccacgccctgct gtgcagctgt 2640 gtgcgttggt gtgtttctgt gtcgctggcg tgtcatgtgatgtagccatg tttgctgaca 2700 tgagcccctg cccccttctc tttttctcca ttggtttctagaactctctt cctcccctcc 2760 cctgagggac aggactcctg gggcctagct gggggcccgagcctggccac cctcctgtta 2820 gccctcagag tcttatttct ctctattggt gaccaagttgcaaatggata aaatacagga 2880 aattctgacc ccctgcccca gacctgcatg tcctgtccccagtgcccccg aaccccatcc 2940 tgggccgggt tgggcctggt gggacgggag aaatagcaactaatccaaca gcgaaaaaaa 3000 aaa 3003 6 825 DNA Rattus norvegicus 6atgcgggacc ggctgccgga cctcacggcg tgtaggaaaa gcgacgatgg agacaatgct 60gtcatcatca cggtggagaa ggaccacttc atggatgcct tcttccatca ggtggaggag 120attcgaagca gcatagccag gattgctcag cacgtggagg atgtgaagaa gaaccacagc 180atcatcctct ctgccccaaa cccagaagga aaaataaaag aagagctgga ggacctgaac 240aaagagatca agaaaactgc taacaggatc cggggcaagc tgaaggctat tgagcagagt 300tgtgatcagg acgagaatgg gaaccgaact tcagtggatc tgcggatacg aaggacccag 360cactcagtgc tgtcacggaa gtttgtggac gtcatgacag aatacaatga agcacagatc 420ctgtttcggg agcgaagcaa aggccgaatc cagcgccagc tagagatcac tgggaggacc 480accactgacg aggagctgga ggagatgctg gagagcggga agccgtccat cttcatctcg 540gacattatat cagattcaca gattactagg caagctctca atgagatcga gtcacgccac 600aaagacatca tgaagctgga gaccagcatc cgagagctgc acgagatgtt catggatatg 660gccatgtttg tcgagactca gggtgaaatg gtcaacaaca ttgagagaaa cgtggtgaac 720tccgtagatt acgtggagca cgccaaggaa gagactaaga aagccatcaa ataccagagc 780aaggccagac ggggtgtgct ctgtgctctc ggccgacagt gctga 825 7 870 DNA Rattusnorvegicus 7 atgcgggacc ggctgccgga cctcacggcg tgtaggaaaa gcgacgatggagacaatgct 60 gtcatcatca cggtggagaa ggaccacttc atggatgcct tcttccatcaggtggaggag 120 attcgaagca gcatagccag gattgctcag cacgtggagg atgtgaagaagaaccacagc 180 atcatcctct ctgccccaaa cccagaagga aaaataaaag aagagctggaggacctgaac 240 aaagagatca agaaaactgc taacaggatc cggggcaagc tgaaggctattgagcagagt 300 tgtgatcagg acgagaatgg gaaccgaact tcagtggatc tgcggatacgaaggacccag 360 cactcagtgc tgtcacggaa gtttgtggac gtcatgacag aatacaatgaagcacagatc 420 ctgtttcggg agcgaagcaa aggccgaatc cagcgccagc tagagatcactgggaggacc 480 accactgacg aggagctgga ggagatgctg gagagcggga agccgtccatcttcatctcg 540 gacattatat cagattcaca gattactagg caagctctca atgagatcgagtcacgccac 600 aaagacatca tgaagctgga gaccagcatc cgagagctgc acgagatgttcatggatatg 660 gccatgtttg tcgagactca gggtgaaatg gtcaacaaca ttgagagaaacgtggtgaac 720 tccgtagatt acgtggagca cgccaaggaa gagactaaga aagccatcaaataccagagc 780 aaggccagac ggaaggtgat gttcgtcctc atctgtgtag tcactttgcttgtgatcctt 840 ggaattattc tagcaacagc attgtcatag 870 8 1109 DNA Homosapiens 8 gcggggcctg aggcggagac cggagagccc gcggcccggc cggaggcagctcgggacagg 60 cttgagcggc ggggcgcgct gcccggccgg cggggatgcg ggaccggctgccagacctga 120 cggcgtgtag gaagaatgat gatggagaca cagttgttgt ggttgagaaagatcatttca 180 tggatgattt cttccatcag gtggaggaga ttagaaacag tattgataaaataactcaat 240 atgttgaaga agtaaagaaa aaccacagca tcattctttc tgcaccaaacccggaaggaa 300 aaataaaaga agagcttgaa gatctgaaca aagaaatcaa gaaaactgcgaataaaattg 360 cagccaagtt aaaggctatt gaacaaagtt ttgatcagga tgagagtgggaaccggactt 420 cagtggatct tcggatacga agaacccagc attcggtgct gtctcggaagtttgtggaag 480 ccatggcgga gtacaatgag gcacagactc tgtttcggga gcggagcaaaggccgcatcc 540 agcgccagct ggagataact gggagaacca ccacagacga cgagctagaagagatgctgg 600 agagcgggaa gccatccatc ttcacttccg acattatatc agattcacaaattactagac 660 aagctctcaa tgaaatcgag tcacgtcaca aggacatcat gaagctggagaccagcatcc 720 gagagttgca tgagatgttc atggacatgg ctatgtttgt ggagactcagggtgaaatga 780 tcaacaacat agaaagaaat gttatgaatg ccacagacta tgtagaacacgctaaagaag 840 aaacaaaaaa agctatcaaa tatcagagca aggcaagaag gaaaaagtggataattattg 900 ctgtgtcagt ggttctggtt gtctatcgtc tatttggctt gtcgttggaatatgttgtac 960 gcagtgctgc ctctctgcca gggtggggaa attgatgttc attatattgaagtttgttta 1020 ttgattctca cacatcaaac caccaagatt cctgctgcaa tgaaccaaatcagcatcctg 1080 tcatttcgtg aatgaatctc agacgctgt 1109 9 288 PRT Rattusnorvegicus 9 Met Arg Asp Arg Leu Pro Asp Leu Thr Ala Cys Arg Lys Ser AspAsp 1 5 10 15 Gly Asp Asn Ala Val Ile Ile Thr Val Glu Lys Asp His PheMet Asp 20 25 30 Ala Phe Phe His Gln Val Glu Glu Ile Arg Ser Ser Ile AlaArg Ile 35 40 45 Ala Gln His Val Glu Asp Val Lys Lys Asn His Ser Ile IleLeu Ser 50 55 60 Ala Pro Asn Pro Glu Gly Lys Ile Lys Glu Glu Leu Glu AspLeu Asn 65 70 75 80 Lys Glu Ile Lys Lys Thr Ala Asn Ile Arg Gly Lys LeuLys Ala Ile 85 90 95 Glu Gln Ser Cys Asp Gln Asp Glu Asn Gly Asn Arg ThrSer Val Asp 100 105 110 Leu Arg Ile Arg Arg Thr Gln His Ser Val Leu SerArg Lys Phe Val 115 120 125 Asp Val Met Thr Glu Tyr Asn Glu Ala Gln IleLeu Phe Arg Glu Arg 130 135 140 Ser Lys Gly Arg Ile Gln Arg Gln Leu GluIle Thr Gly Arg Thr Thr 145 150 155 160 Asp Glu Glu Leu Glu Glu Met LeuGlu Ser Gly Lys Pro Ser Ile Phe 165 170 175 Ile Ser Asp Ile Ile Ser AspSer Gln Ile Thr Arg Gln Ala Leu Asn 180 185 190 Glu Ile Glu Ser Arg HisLys Asp Ile Met Lys Leu Glu Thr Ser Ile 195 200 205 Arg Glu Leu His GluMet Phe Met Asp Met Ala Met Phe Val Glu Thr 210 215 220 Gln Gly Glu MetVal Asn Asn Ile Glu Arg Asn Val Val Asn Ser Val 225 230 235 240 Asp TyrVal Glu His Ala Lys Glu Glu Thr Lys Lys Ala Ile Lys Tyr 245 250 255 GlnSer Lys Ala Arg Arg Lys Lys Trp Ile Ile Ala Ala Val Val Val 260 265 270Ala Val Ile Ala Val Leu Ala Leu Ile Ile Gly Leu Thr Val Gly Lys 275 280285 10 287 PRT Rattus norvegicus 10 Met Arg Asp Arg Leu Pro Asp Leu ThrAla Cys Arg Lys Ser Asp Asp 1 5 10 15 Gly Asp Asn Ala Val Ile Ile ThrVal Glu Lys Asp His Phe Met Asp 20 25 30 Ala Phe Phe His Gln Val Glu GluIle Arg Ser Ser Ile Ala Arg Ile 35 40 45 Ala Gln His Val Glu Asp Val LysLys Asn His Ser Ile Ile Leu Ser 50 55 60 Ala Pro Asn Pro Glu Gly Lys IleLys Glu Glu Leu Glu Asp Leu Asn 65 70 75 80 Lys Glu Ile Lys Lys Thr AlaAsn Ile Arg Gly Lys Leu Lys Ala Ile 85 90 95 Glu Gln Ser Cys Asp Gln AspGlu Asn Gly Asn Arg Thr Ser Val Asp 100 105 110 Leu Arg Ile Arg Arg ThrGln His Ser Val Leu Ser Arg Lys Phe Val 115 120 125 Asp Val Met Thr GluTyr Asn Glu Ala Gln Ile Leu Phe Arg Glu Arg 130 135 140 Ser Lys Gly ArgIle Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr 145 150 155 160 Asp GluGlu Leu Glu Glu Met Leu Glu Ser Gly Lys Pro Ser Ile Phe 165 170 175 IleSer Asp Ile Ile Ser Asp Ser Gln Ile Thr Arg Gln Ala Leu Asn 180 185 190Glu Ile Glu Ser Arg His Lys Asp Ile Met Lys Leu Glu Thr Ser Ile 195 200205 Arg Glu Leu His Glu Met Phe Met Asp Met Ala Met Phe Val Glu Thr 210215 220 Gln Gly Glu Met Val Asn Asn Ile Glu Arg Asn Val Val Asn Ser Val225 230 235 240 Asp Tyr Val Glu His Ala Lys Glu Glu Thr Lys Lys Ala IleLys Tyr 245 250 255 Gln Ser Lys Ala Arg Arg Lys Val Met Phe Val Leu IleCys Val Val 260 265 270 Thr Leu Leu Val Ile Leu Gly Ile Ile Leu Ala ThrAla Leu Ser 275 280 285 11 272 PRT Rattus norvegicus 11 Met Arg Asp ArgLeu Pro Asp Leu Thr Ala Cys Arg Lys Ser Asp Asp 1 5 10 15 Gly Asp AsnAla Val Ile Ile Thr Val Glu Lys Asp His Phe Met Asp 20 25 30 Ala Phe PheHis Gln Val Glu Glu Ile Arg Ser Ser Ile Ala Arg Ile 35 40 45 Ala Gln HisVal Glu Asp Val Lys Lys Asn His Ser Ile Ile Leu Ser 50 55 60 Ala Pro AsnPro Glu Gly Lys Ile Lys Glu Glu Leu Glu Asp Leu Asn 65 70 75 80 Lys GluIle Lys Lys Thr Ala Asn Ile Arg Gly Lys Leu Lys Ala Ile 85 90 95 Glu GlnSer Cys Asp Gln Asp Glu Asn Gly Asn Arg Thr Ser Val Asp 100 105 110 LeuArg Ile Arg Arg Thr Gln His Ser Val Leu Ser Arg Lys Phe Val 115 120 125Asp Val Met Thr Glu Tyr Asn Glu Ala Gln Ile Leu Phe Arg Glu Arg 130 135140 Ser Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr 145150 155 160 Asp Glu Glu Leu Glu Glu Met Leu Glu Ser Gly Lys Pro Ser IlePhe 165 170 175 Ile Ser Asp Ile Ile Ser Asp Ser Gln Ile Thr Arg Gln AlaLeu Asn 180 185 190 Glu Ile Glu Ser Arg His Lys Asp Ile Met Lys Leu GluThr Ser Ile 195 200 205 Arg Glu Leu His Glu Met Phe Met Asp Met Ala MetPhe Val Glu Thr 210 215 220 Gln Gly Glu Met Val Asn Asn Ile Glu Arg AsnVal Val Asn Ser Val 225 230 235 240 Asp Tyr Val Glu His Ala Lys Glu GluThr Lys Lys Ala Ile Lys Tyr 245 250 255 Gln Ser Lys Ala Arg Arg Gly ValLeu Cys Ala Leu Gly Arg Gln Cys 260 265 270 12 299 PRT Homo sapiens 12Met Arg Asp Arg Leu Pro Asp Leu Thr Ala Cys Arg Lys Asn Asp Asp 1 5 1015 Gly Asp Thr Val Val Val Val Glu Lys Asp His Phe Met Asp Asp Phe 20 2530 Phe His Gln Val Glu Glu Ile Arg Asn Ser Ile Asp Lys Ile Thr Gln 35 4045 Tyr Val Glu Glu Val Lys Lys Asn His Ser Ile Ile Leu Ser Ala Pro 50 5560 Asn Pro Glu Gly Lys Ile Lys Glu Glu Leu Glu Asp Leu Asn Lys Glu 65 7075 80 Ile Lys Lys Thr Ala Asn Lys Ile Ala Ala Lys Leu Lys Ala Ile Glu 8590 95 Gln Ser Phe Asp Gln Asp Glu Ser Gly Asn Arg Thr Ser Val Asp Leu100 105 110 Arg Ile Arg Arg Thr Gln His Ser Val Leu Ser Arg Lys Phe ValGlu 115 120 125 Ala Met Ala Glu Tyr Asn Glu Ala Gln Thr Leu Phe Arg GluArg Ser 130 135 140 Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly ArgThr Thr Thr 145 150 155 160 Asp Asp Glu Leu Glu Glu Met Leu Glu Ser GlyLys Pro Ser Ile Phe 165 170 175 Thr Ser Asp Ile Ile Ser Asp Ser Gln IleThr Arg Gln Ala Leu Asn 180 185 190 Glu Ile Glu Ser Arg His Lys Asp IleMet Lys Leu Glu Thr Ser Ile 195 200 205 Arg Glu Leu His Glu Met Phe MetAsp Met Ala Met Phe Val Glu Thr 210 215 220 Gln Gly Glu Met Ile Asn AsnIle Glu Arg Asn Val Met Asn Ala Thr 225 230 235 240 Asp Tyr Val Glu HisAla Lys Glu Glu Thr Lys Lys Ala Ile Lys Tyr 245 250 255 Gln Ser Lys AlaArg Arg Lys Lys Trp Ile Ile Ile Ala Val Ser Val 260 265 270 Val Leu ValVal Tyr Arg Leu Phe Gly Leu Ser Leu Glu Tyr Val Val 275 280 285 Arg SerAla Ala Ser Leu Pro Gly Trp Gly Asn 290 295

What is claimed:
 1. A composition comprising: an effective amount of an inhibitor of a SNARE protein, said inhibitor effective in preventing cytokinesis in a population of cells.
 2. The composition of claim 1 wherein said SNARE protein inhibitor is expressed in said cells.
 3. The composition of claim 1 wherein said dominant-negative inhibitor is expressed using an virus vector with a regulatable promotor.
 4. The composition of claim 1 wherein said dominant-negative inhibitor is expressed using an adenovirus vector with a tetracycline-regulatable promotor.
 5. The composition of claim 1 wherein said SNARE protein inhibitor comprises a SNARE protein antibody
 6. The composition of claim 1 wherein said inhibitor induces expression of a soluble isoforms of syntaxin
 2. 7. The composition of claim 1, wherein said inhibitor induces expression of a soluble isoforms of endobrevin.
 8. The composition of claim 6, wherein said soluble isoform of syntaxin 2 is selected from the group consisting of Syntaxin 2C and Syntaxin 2D.
 9. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.
 10. A method of treating, preventing, or ameliorating a disease in a patient in need thereof comprising: administering to said pateint an effective amount of a composition effective in inhibiting SNARE function during cytokinesis.
 11. The method of claim 10 wherein said composition comprises antibodies of SNARE proteins.
 12. The method of claim 10 wherein said composition comprises antibodies of syntaxin
 2. 13. The method of clam 10 wherein said composition comprises antibodies of endobrevin.
 14. The method of claim 10 wherein said composition forms nonfunctional complexes with SNARE proteins. 15 The method of claim 10 wherein said composition is administered to said patient to induce over expression of syntaxin 2 isoforms in said patient.
 16. The method of claim 10 wherein said composition is administered to said patient to induce over expression of endobrevin isoforms in said patient.
 17. A method of forming binucleated cells comprising: administering to a collection of cells an effective amount of a composition to form binucleated cells from cells comprising said collection of cells, said composition comprising an inhibitor of a SNARE protein.
 18. The method of claim 9 wherein said composition comprises a SNARE protein inhibitor.
 19. The method of claim 10 wherein said composition comprises a SNARE protein inhibitor chosen from the group consisting of antibodies, DNA constructs, toxins, competing analogs of SNARE proteins, and combinations of any of these.
 20. The method of claim 17, wherein said binucleated cells formed thereby are more susceptible to apoptosis. 